OPEN
Citation: Cell Death and Disease (2013) 4, e661; doi:10.1038/cddis.2013.179
& 2013 Macmillan Publishers Limited All rights reserved 2041-4889/13
www.nature.com/cddis
Cell cycle-arrested tumor cells exhibit increased
sensitivity towards TRAIL-induced apoptosis
H Ehrhardt1,2,4, F Wachter1,4, M Grunert1 and I Jeremias*,1,3
Resting tumor cells represent a huge challenge during anticancer therapy due to their increased treatment resistance.
TNF-related apoptosis-inducing ligand (TRAIL) is a putative future anticancer drug, currently in phases I and II clinical studies.
We recently showed that TRAIL is able to target leukemia stem cell surrogates. Here, we tested the ability of TRAIL to target cell
cycle-arrested tumor cells. Cell cycle arrest was induced in tumor cell lines and xenografted tumor cells in G0, G1 or G2 using
cytotoxic drugs, phase-specific inhibitors or RNA interference against cyclinB and E. Biochemical or molecular arrest at any
point of the cell cycle increased TRAIL-induced apoptosis. Accordingly, when cell cycle arrest was disabled by addition of
caffeine, the antitumor activity of TRAIL was reduced. Most important for clinical translation, tumor cells from three children with
B precursor or T cell acute lymphoblastic leukemia showed increased TRAIL-induced apoptosis upon knockdown of either
cyclinB or cyclinE, arresting the cell cycle in G2 or G1, respectively. Taken together and in contrast to most conventional
cytotoxic drugs, TRAIL exerts enhanced antitumor activity against cell cycle-arrested tumor cells. Therefore, TRAIL might
represent an interesting drug to treat static-tumor disease, for example, during minimal residual disease.
Cell Death and Disease (2013) 4, e661; doi:10.1038/cddis.2013.179; published online 6 June 2013
Subject Category: Cancer
Resting tumor cells exhibit a severe challenge during anticancer treatment. In minimal residual disease, surviving tumor
cells might stay quiescent and induce relapse after a
prolonged period of time. Resting tumor cells display
enhanced treatment resistance compared with actively
cycling tumor cells, as several groups of anticancer drugs
directly target the active cell cycle. Cancer stem cells are
known to remain resting. To increase the prognosis and cure
rate of cancer, anticancer therapy has to remove resting tumor
cells.1–4
TNF-related apoptosis-inducing ligand (TRAIL) is a promising future anticancer drug due to its tumor selectivity, almost in
the absence of side effects in animal trials, and phases I and II
clinical studies.5,6 The intracellular apoptosis signal transduction initiated by TRAIL is well characterized and involves the
TRAIL-death receptors, FADD (Fas-associated protein with
death domain) as adapter protein and caspases. The
apoptosis signal of TRAIL might be amplified by mitochondria,
which is regulated by members of the Bcl-2 family. Further
regulators of TRAIL-induced apoptosis are the Caspase-8
antagonist FLIP (FLICE inhibitory protein) and members of
the IAP-family including XIAP, which antagonize downstream
caspases.7–9
Although the antitumor effect of TRAIL as a single agent is
limited, TRAIL exerts remarkable antitumor activity upon
combination with established cytotoxic drugs in phases I and II
clinical trials. Cytotoxic drugs like doxorubicin (doxo) or
methotrexate (MTX) and others induce synergistic apoptosis
upon combination with TRAIL.7,8,10–14
Several different mechanisms have been described of how
cytotoxic drugs sensitize tumor cells towards TRAIL-induced
apoptosis. Among them, the transcription factor p53 is
activated by several established cytotoxic drugs and mediates
a number of different effects in tumor cells including gene
regulation, apoptosis and cell cycle arrest.15,16 As several
proteins mediating or regulating TRAIL-induced apoptosis are
p53 target genes, for example, TRAIL receptor 2 (death
receptor 5, DR5), p53-mediated gene regulation is suggested
to be the main mechanism for mediating synergistic apoptosis
of cytotoxic drugs and TRAIL.6,9,14,17
We have recently described the importance of p53mediated cell cycle arrest for inhibiting vinca alkaloid-induced
apoptosis.18 We also described that TRAIL damages stem
cell surrogates in patient-derived leukemia cells.19 As cancer
stem cells are often resting, we hypothesized that TRAIL
might be able to induce apoptosis in resting tumor cells.
Although chemical compounds or drugs in preclinical testing
were shown to sensitize tumor cells towards TRAIL-induced
apoptosis accompanied by cell cycle arrest,20–22 no molecular
data exist so far and no data on patients’ tumor cells are
present. Therefore, we studied here how cell cycle arrest
influences the ability of TRAIL to induce apoptosis in
tumor cells, using molecular approaches in patient-derived
tumor cells.
1
Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany; 2Division of Neonatology, Perinatal Center, University Children’s
Hospital, Ludwig-Maximilians-University Munich, Munich, Germany and 3Department of Oncology/Hematology, Dr. von Haunersches Kinderspital, München, Germany
*Corresponding author: I Jeremias, Helmholtz Zentrum München, German Research Center for Environmental Health, Marchioninistrasse 25, München D-81377,
Germany. Tel: +49 89 7099 424; Fax: +49 89 7099 225; E-mail: [email protected]
4
These authors contributed equally to this work.
Keywords: cell cycle arrest; TRAIL; apoptosis; DR5; cyclins
Abbreviation: ALL, acute lymphatic leukemia; Bcl-2, B-cell lymphoma 2; Dexa, Dexamethasone; Doxo, Doxorubicin; FADD, Fas-associated protein with death domain;
FLIP, FLICE inhibitory protein; IAP, suppressors of apoptosis; MTX, Methotrexate; TRAIL, TNF-related apoptosis-inducing ligand; XIAP, X-linked inhibitor of apoptosis
Received 13.12.12; revised 15.3.13; accepted 12.4.13; Edited by G Ciliberto
Increased activity of TRAIL against cell cycle-arrested tumor cells
H Ehrhardt et al
2
Results
Several cytotoxic drugs sensitize towards TRAIL-induced
apoptosis and induce p53-typic effects. Numerous
conventional cytotoxic drugs of current clinical routine are
known to induce cell cycle arrest mediated by the transcription
factor p53. In a first approach, cell cycle arrest was induced
using cytotoxic drugs.
We used SHEP neuroblastoma cells (printed Figures 1–4)
and HCT116 colon cancer cells (Supplementary Figures S2,
S4 and S5), both of which express functionally active p53 in
wild-type conformation.23 Additionally, CEM T-ALL leukemia
cells were included with mutant, but functionally active, p53
(Supplementary Figure S6)11,18,23,24 and xenografted ALL
leukemia samples (Figures 5 and 6 and Supplementary
Figure S7). As described for several cell lines in the
literature,12–14 both SHEP and HCT116 cells displayed
prominent synergistic apoptosis induction when TRAIL was
combined with doxo (Figure 1a and Supplementary Figure
S2A). Apoptosis data were congruent with increased caspase
cleavage for the drug combination (Figure 1b). Concomitantly,
doxo strongly activated p53 in both cell lines. According to the
known different effects of p53, doxo upregulated typical p53
target genes in the TRAIL apoptosis signaling pathway,
mainly TRAIL receptor-2 and Caspase-10, and arrested the
cell cycle (Figures 1c and d, Supplementary Figures S1A
and B, and S2B and C). The upregulation of TRAIL receptor-2
by doxo was higher in cells with cell cycle arrest in G2
(Supplementary Figure S1C). As TRAIL receptor-2 regulation
has been reported to be a central determinant of TRAIL
sensitivity, overexpression of TRAIL receptor-2 was performed, but did not have an impact on the TRAIL response,
arguing against a dominant role of TRAIL receptor-2 expression levels in the regulation of TRAIL sensitivity in our
experimental setting (Supplementary Figure S1D). Determination of the phosphorylation status at Serine 10 of Histone
H3 revealed that the cell cycle was arrested in G2, but not in M
(Figures 1c and d, Supplementary Figures S2D and E). For
the combination of doxo plus TRAIL, the fraction of cells in G2
was markedly reduced (Figure 1e). To prevent the cell cycle
arrest by cytotoxic drugs, the biochemical inhibitor caffeine
was used, as it is highly efficient with nearly absent toxicity and
not specific to a certain phase of the cell cycle or
chemotherapeutic drug applied. Synergistic apoptosis induction was markedly reduced by pretreatment with caffeine,
which also prevented p53 accumulation, reduced the upregulation of TRAIL receptor-2 and Caspase-10, and the cell
cycle arrest by doxo (Figures 1a–d, Supplementary Figures
S1A and B, and S2A-E).
Similarly, MTX and dexamethasone (dexa) induced superadditive apoptosis with TRAIL (Figures 2a and b), and both
drugs arrested the cell cycle in G1 (Figure 2c and data not
shown). Whereas MTX increased the expression of TRAIL
receptor-2, dexa did not relevantly alter the mean fluorescence intensity (Supplementary Figure S3). Pretreatment with
caffeine reduced synergistic apoptosis, TRAIL receptor-2
upregulation and cell cycle arrest (Figures 2a–c,
Supplementary Figure S3 and data not shown).
Taken together, several cytotoxic drugs sensitized towards
TRAIL-induced apoptosis, induced cell cycle arrest at
Cell Death and Disease
different phases and upregulated typical p53 target genes,
which were all inhibited by caffeine.
Cell cycle inhibitors sensitize for TRAIL-induced
apoptosis. Cytotoxic drugs are mainly described to promote
TRAIL-induced apoptosis by the regulation of apoptosis
protein expression including TRAIL receptor-2 expression.6,9,14–17 The data presented so far do not allow estimation
of the specific contribution of cell cycle arrest. Next, we aimed
at discriminating between the different effects of p53 induced
by cytotoxic drugs, and asked whether cell cycle arrest itself
might be sufficient to sensitize towards TRAIL-induced
apoptosis. Towards this aim, we used biochemical cell cycle
inhibitors or irradiation, which are known to arrest cells in
defined phases of the cell cycle. As published before, FCS
withdrawal arrested the cells in G0, mimosine in G1 and
irradiation in G2 (Table 1 and Supplementary Table 1).25
Interestingly, upon arresting the cell cycle in any given phase,
both compounds and irradiation significantly sensitized for
TRAIL-induced apoptosis (Figure 3a). The analysis with
isobolograms showed the synergistic effects clearly
(Figure 3b and Supplementary Figure S4). Taken together,
biochemical cell cycle arrest was associated with particularly
efficient cell death induction by TRAIL.
Knockdown of cyclinB or cyclinE induce cell cycle arrest
and sensitize for TRAIL-induced apoptosis. So far, we
showed that various drugs, compounds and stimuli induced
cell cycle arrest and sensitized towards TRAIL-induced
apoptosis. We next asked whether cell cycle arrest was
mechanistically responsible for sensitizing towards TRAILinduced apoptosis. To discriminate between drug-induced
cell cycle arrest and further drug-induced, p53-mediated
effects such as gene regulation, cell cycle arrest was induced
by molecular manipulation using RNA interference.
Cyclins are regulators of the cell cycle that control transition
through the different phases of the cell cycle. Whereas
expression of cyclinB is a prerequisite for transition from
G2 to M, cyclinE controls the slip from G1 to S-phase. Using
RNA interference, we studied the impact of downregulation of
these cyclines on TRAIL-induced apoptosis.
According to published data, knockdown of cyclinB or
cyclinE induced cell cycle arrest in G2 or G1 (Figures 4a–c
and Supplementary Figure S5A). As published for the cell cycle
arrest in G1 by inhibition of cyclinD1 before, we preferred an
siRNA sequence against cyclinE that leads to an incomplete,
but statistically significant cell cycle arrest in G1, but does not
affect the basal apoptosis rate of transfected cells.25–27 The
expression levels of the apoptosis signaling proteins studied
and p53 remained unchanged in cells with knockdown of
cyclinB or cyclinE. Of special interest, the expression level of
TRAIL receptor-1 and -2 remained unchanged (Figures 4d and
e). Knockdown of cyclinB or cyclinE significantly sensitized the
solid tumor cell lines SHEP and HCT116 for apoptosis induction
by TRAIL (Figure 4f and Supplementary Figure S5B). In line
with our previous results for vincristine-induced apoptosis, the
slight alteration of cells in G1 was associated with a marked
difference in apoptosis induction by TRAIL.25 Dose–response
curves confirmed the general impact of knockdown of cyclinB
or E on TRAIL-induced apoptosis (Figure 4g).
Increased activity of TRAIL against cell cycle-arrested tumor cells
H Ehrhardt et al
3
SHEP
SHEP
untreated
TRAIL
doxo
doxo+TRAIL
untreated
*
doxo
500
*
400
NS
events
events
specific apoptosis (%)
600
50
400
16.9%
200
25
300
200
56.0%
100
0
0
50
0
100 150 200
propidium iodide
0
250 k
50
100 150 200
propidium iodide
250 k
0
– caffeine
+ caffeine
caffeine
caffeine+doxo
600
600
SHEP
co TRAIL doxo doxo co TRAIL doxo doxo
+TRAIL
+TRAIL
events
events
+ caffeine
– caffeine
400
cl. Casp-3
200
200
GAPDH
400
16.5%
31.1%
0
0
50
0
100 150 200
propidium iodide
250 k
14.8%
102
0
600
450
0.1%
103
55.9%
600
events
104
300
102
300
19.0%
20.5%
150
0
0
50
100 150 200
propidium iodide
250 k
0
caffeine
50 100 150 200
propidium iodide
250 k
0
0
105
103
14.8%
102
250 k
0
104
50
100 150 200
propidium iodide
250 k
doxo+TRAIL
600
150
450
0.2%
events
1.7%
p-HistoneH3 Ser10
104
100 150 200
propidium iodide
doxo
caffeine+doxo
105
50
103
events
0
p-HistoneH3 Ser10
250 k
TRAIL
900
events
103
p-HistoneH3 Ser10
p-HistoneH3 Ser10
105
2.1%
100 150 200
propidium iodide
untreated
doxo
104
50
SHEP
SHEP
untreated
105
0
300
40.8%
30.9%
102
100
0.8%
50
150
0
0
0
0
50
100 150 200
propidium iodide
250 k
0
50 100 150 200
propidium iodide
250 k
0
0
50
100 150 200
propidium iodide
250 k
0
50
100 150 200
propidium iodide
250 k
Figure 1 Super-additive activity of doxo and TRAIL in SHEP cells. (a) SHEP cells were pretreated with caffeine (300 mg/ml) for 12 h or left untreated, followed by stimulation
with doxo (100 ng/ml) for 48 h. TRAIL (100 ng/ml) was added afterwards for another 24 h. (b) SHEP cells pretreated and stimulated as in(a) were analyzed for caspase-3
cleavage. co ¼ untreated, cl. Casp-3 ¼ cleaved Caspase-3. (c, d) SHEP cells treated with caffeine and doxo as in (a) were analyzed for cell cycle distribution using propidium
iodide staining (c). G2 and M-phase were discriminated by the simultaneous staining for p-HistoneH3Ser10 (d). (e) SHEP cells were stimulated with doxo (30 ng/ml) for 48 h,
followed by TRAIL (10 ng/ml). Cell cycle analysis was performed 24 h after the addition of TRAIL using propidium iodide staining, as in (c), gating on living cells. Cell death
induction was measured by Nicoletti staining. Statistical analysis was performed comparing the apoptosis induction of the combined stimulation to the addition of cell death
induction by doxo and TRAIL alone, and comparing the combinatorial application of pretreated and untreated cells with paired t-test. *Po0,05, NS ¼ statistically not significant
To prove the general significance of the observed phenotype across different tumor entities, the hematopoietic T cell
leukemia cell line CEM was additionally studied, which
expresses mutant but functionally active p53.11,18,23,24,28
siRNA against cyclinB and E arrested the cell cycle in G2
and G1, respectively, also to a minor extent compared with the
Cell Death and Disease
Increased activity of TRAIL against cell cycle-arrested tumor cells
H Ehrhardt et al
4
SHEP
100
untreated
TRAIL
MTX
MTX+TRAIL
*
SHEP
*
untreated
75
NS
300
events
200
50
events
specific apoptosis (%)
MTX
300
400
200
18.6%
25
0
0
control
0
0
caffeine
50
SHEP
untreated
TRAIL
dexa
dexa+TRAIL
100
150
200
propidium iodide
250 k
0
50
caffeine
*
100
150
200
propidium iodide
250 k
caffeine+MTX
*
400
400
50
NS
300
300
events
events
specific apoptosis (%)
9.7%
100
100
200
19.9%
21.0%
25
200
100
100
0
0
0
50
100
150
propidium iodide
200
250 k
0
50
100
150
200
250 k
propidium iodide
0
control
caffeine
Figure 2 Augmented apoptosis-inducing capacity of TRAIL associated with cell cycle arrest by MTX and dexamethasone. (a, b) SHEP cells were pretreated with caffeine
for 12 h, followed by stimulation with methotrexate (30 mM; (a) or dexamethasone (10 5 M; (b) for 48 h. TRAIL (100 ng/ml) was added afterwards for another 24 h. (c) SHEP
cells treated with caffeine and MTX ,as in (a), were analyzed, as in Figure 1b. Determination of apoptosis induction, presentation and analysis of the data and statistical
analysis were performed as in Figure 1. *Po0.05
solid tumor cell lines (Supplementary Figure S6A). Similar to
the solid tumor cell lines studied, cell cycle arrest sensitized
towards TRAIL-induced apoptosis in CEM cells
(Supplementary Figure S6B).
These data show that cell cycle arrest itself sensitizes
towards TRAIL-induced apoptosis in the clear absence of
protein regulation.
Patient-derived tumor cells are sensitized for
TRAIL-induced apoptosis by cytotoxic drugs. Established
cell lines might have acquired additional, non-physiologic
mutations upon prolonged culture in vitro. For example, the
majority of leukemic cell lines inherit mutations in p53, which
are rarely found in leukemia patients.23,29–31 To exclude a
culture-specific artifact and to go beyond cell line work, tumor
cells from patients were studied. Towards this aim, we used
primary tumor cells from children with acute leukemia. As
these cells are notoriously reluctant towards in vitro growth,
primary cells were passaged through immunocompromised
mice,11,32 where they remain largely genetically stable.33
Three different ALL samples were stimulated with doxo and
TRAIL, with and without pretreatment with caffeine. Whereas
Cell Death and Disease
doxo partially arrested the cells in G2, caffeine markedly
reduced the G2 arrest (Figure 5a and Supplementary Figure
S7A). On a functional level and in accordance to data obtained
in cell lines, doxo and TRAIL induced synergistic apoptosis,
which was inhibited by pretreatment with caffeine (Figure 5b
and Supplementary Figures S7B and C).
Patient-derived tumor cells are sensitized towards
TRAIL-induced apoptosis by knockdown of cyclinB or
cyclinE. To prove that cell cycle arrest was capable to
sensitize towards TRAIL-induced apoptosis, patient-derived
ALL cells were transfected with siRNA targeting cyclinB or E,
using our recently described technique.11,24,32 Whereas
siRNA against cyclinB accumulated cells in G2, siRNA
against cyclinE increased the fraction of cells in G1
(Figure 6a and data not shown). Concomitantly, knockdown
of either cyclinB or cyclinE augmented TRAIL-induced
apoptosis in ALL cells of all three patients (Figure 6b and
Supplementary Figures S7D and E).
Thus, cell cycle arrest augmented TRAIL-induced apoptosis not only in cell line cells, but also in tumor cells derived from
various children with B precursor ALL. Taken together and in
Increased activity of TRAIL against cell cycle-arrested tumor cells
H Ehrhardt et al
5
SHEP
specific apoptosis (%)
*
50
*
*
25
0
TRAIL
0%FCS
mimosine
irradiation
+
-
+
-
+
+
-
+
-
+
+
-
+
+
+
SHEP
1.0
0.75
0.75
0.75
0.5
0.25
TRAIL
1.0
TRAIL
TRAIL
SHEP
1.0
0.5
0.25
0.25
0
0
0
0
0.25
0.5
0.75
1.0
0.5
0
0.25
FCS withdrawel
0.5
mimosine
0.75
1.0
0
0.25
0.5
0.75
1.0
irradiation
Figure 3 Biochemical cell cycle arrest sensitizes for TRAIL. (a) SHEP cells were pretreated with FCS withdrawal, preincubated with mimosine (100 mM) or irradiated with
30 Gy for 24 h. TRAIL (100 ng/ml) was added for another 24 h. (b) Isobolograms were applied to the experimental setting from (a) to test for synergistic effects of FCS
withdrawal (0, 0,1 and 1% FCS), mimosine (10, 30 and 100 mM) treatment or irradiation (3, 6 and 10 Gy) plus TRAIL (3, 10 and 30 ng/ml). Determination of apoptosis induction,
presentation and analysis of the data, and statistical analysis were performed as in Figure 1. Each data point represents the dose-equation of the applied combinatorial
treatment. *Po0.05
Table 1 Cell cycle distribution in SHEP cells after cell cycle inhibition
Cell cycle distribution
Control
0% FCS
Mimosine
Irradiation
G0 (%)
0.2±0.2
30.9±0.9
0.1±0.1
1.1±0.3
G1 (%)
G2 (%)
M (%)
67.8±3.6 18.8±4.5 2.8±0.8
52.6±2.9
9.8±0.5 0.2±0.1
85.0±3.5 10.9±1.0 0.1±0.1
15.8±2.3 75.2±3.8
0
SHEP cells were treated by withdrawal of FCS (0% FCS), with mimosine
(100 mM) or irradiated (30 Gy) for 24 h. Cell cycle distribution was analyzed with
propidium iodide in combination with cyclinD1 staining to discriminate G0 and
G1 phases, and with p-Histone H3 staining to separate the arrest in G2 and
M-phase. Data are presented as mean±S.E.M. of three independent
experiments.
contrast to conventional chemotherapeutics, TRAIL induces
apoptosis more efficiently in tumor cells during cell cycle
arrest compared with actively cycling tumor cells.
Discussion
Our data show that TRAIL induces apoptosis more efficiently if
tumor cells undergo cell cycle arrest compared with actively
cycling tumor cells. For the first time, we obtained mechanistic
proof that cell cycle arrest itself sensitizes tumor cells towards
TRAIL-induced apoptosis, including patients’ tumor cells. This
finding was obtained by inducing cell cycle arrest by
(i) conventional cytotoxic drugs; (ii) known cell cycle arrestors
or (iii) molecularly by knockdown of certain cyclines. Knockdown-induced cell cycle arrest sensitized towards TRAIL-
induced apoptosis in cell lines of various different tumor
entities, as well as in patient-derived leukemia cells.
Therapeutic targeting of cells in cell cycle arrest is of high
clinical importance. Cancer stem cells are known for their low
cycling activity and chemoresistance. Static-tumor diseases
are especially difficult to treat, for example, during minimal
residual disease or in low-grade tumors. Insufficient treatment
of static-tumor disease often results in tumor relapse. Our
finding might suggest testing TRAIL in static-tumor disease
in vivo as TRAIL seems to be especially efficient against
resting tumor cells.
As TRAIL induces limited apoptosis in most primary tumor
cells when given alone, the combined use of TRAIL together
with conventional cytotoxic drugs has been intensively studied
over the last years. Several different conventional anticancer
drugs strongly sensitize tumor cells towards TRAIL-induced
apoptosis. In search for underlying signaling mechanisms,
p53 and its downstream effects were studied intensively.
Most cytotoxic drugs accumulate and activate p53. p53mediated gene regulation of signaling mediators of TRAILinduced apoptosis such as TRAIL receptor-2 was thought
to be responsible for drug-induced sensitization towards
TRAIL-induced apoptosis. These considerations were
used to optimize combinatorial approaches involving
TRAIL.6,8,9,14,17,34
Besides protein regulations, p53 induces cell cycle arrest.
Although p53 is mutated in many tumor cells, leading to altered
p53 function, induction of cell cycle arrest is not affected by
loss of DNA-binding capacity in most p53 mutants.34,35 Our
Cell Death and Disease
Increased activity of TRAIL against cell cycle-arrested tumor cells
H Ehrhardt et al
6
SHEP
SHEP
100
100
mock
sicyclinB
*
100
100
60
75
40
20
20
0
0
0
50
100 150 200
propidium iodide
250 k
0
50 100 150 200
propidium iodide
75
cells in G2 (%)
40
*
cells in G1 (%)
events (%)
60
*
*
80
80
events (%)
mock
sicyclinE
50
25
250 k
50
25
mock shcyclinB shcyclinE
cyclinB
0
mock
cyclinE
0
shcyclinB shcyclinE
mock
shcyclinB shcyclinE
GAPDH
SHEP
isotype
mock
shcyclinB
isotype
mock
shcyclinB
100
100
80
80
60
60
NOXA
Caspase-10
PUMA
FADD
FLIPL
Mcl-1
events
mock shcyclinB shcyclinE
mock shcyclinB shcyclinE
Caspase-8
events
SHEP
40
40
Bax
FLIPS
20
20
Bak
GAPDH
Bid
0
2
0 10
Bcl-2
Bcl-XL
103
104
DR4-Alx647
0
105
0 102
isotype
mock
shcyclinE
XIAP
cIAP-1
103
104
DR5-Alx647
105
isotype
mock
shcyclinE
100
100
80
80
60
60
cIAP-2
p53
events
events
GAPDH
40
20
20
0
0 102
103
104
DR4-Alx647
SHEP
specific apoptosis (%)
specific apoptosis (%)
25
+
–
+
shcyclinB
2
0 10
103
104
DR5-Alx647
105
*
mock
shcyclinB
shcyclinE
50
mock
0
SHEP
*
–
105
100
*
0
TRAIL
40
–
+
shcyclinE
75
*
*
50
25
*
0
0
1
10
TRAIL (ng/ml)
100
Figure 4 Molecular cell cycle arrest sensitizes for TRAIL. (a) SHEP cells transfected with shRNA against cyclinB or cyclinE were analyzed for the cell cycle distribution, as
in Figure 1c. (b–e) SHEP cells from (a) were analyzed for cell cycle distribution in G1 (b) and G2 (c), apoptosis protein expression (d) and TRAIL-death receptor-1 and -2
expression (e) using the experimental setting from Figures 1c and d and Supplementary Figures 1A and 1B. (f) SHEP cells from (a) were stimulated with TRAIL (10 ng/ml). (g)
TRAIL dose–response curves were performed in parental SHEP cells, and cells with knockdown of cyclinB or cyclinE from (a). Determination of cell cycle distribution and
apoptosis induction, presentation and analysis of the data, and statistical analysis were performed as in Figure 1. For multivariate analysis, RM ANOVA was used. *Po0.05
Cell Death and Disease
Increased activity of TRAIL against cell cycle-arrested tumor cells
H Ehrhardt et al
7
ALL-54
untreated
doxo
1200
900
events
events
900
600
600
14.8%
22.2%
300
300
0
0
0
50
100
150
200
propidium iodide
250 k
caffeine
50
0
100
150
200
propidium iodide
250 k
caffeine+doxo
1500
1500
1200
events
events
1200
900
600
11.7%
900
600
14.0%
300
300
0
0
0
50
100
150
200
propidium iodide
250 k
50
0
100
150
200
propidium iodide
250 k
ALL-54
specific apoptosis (%)
50
untreated
TRAIL
doxo
doxo+TRAIL
*
*
NS
25
0
control
caffeine
Figure 5 Doxo-induced cell cycle arrest associated with efficient TRAIL apoptosis induction in xenografted ALL cells. (a, b) Xenografted pre-B ALL-54 cells were
pretreated with caffeine (100 mg/ml) for 12 h, followed by stimulation with doxo (30 ng/ml). After 24 h of incubation with doxo, cell cycle analysis was performed (a) or cells were
stimulated with TRAIL (10 ng/ml) for another 24 h (b). Determination of cell cycle distribution and apoptosis induction, presentation and analysis of the data and statistical
analysis were performed, as in Figure 1. *Po0.05
data show that in addition to the dominant p53-mediated gene
regulation, p53-mediated cell cycle arrest represents a
mechanism by which cytotoxic drugs sensitize tumor cells
towards TRAIL-induced apoptosis mediated by p53.
We have recently described that anthracyclines and vinca
alkaloids are less effective when applied simultaneously as
anthracyclines induce cell cycle arrest, whereas vinca
alkaloids require active cell cycling for antitumor efficiency.18
In contrast, cell cycle arrest is beneficial for TRAIL. The data
presented here widen the therapeutic potential for TRAIL to all
phases of the cell cycle. Our data add to the controversial
discussion, whether or when cell cycle arrest is beneficial,
irrelevant or detrimental during anticancer therapy, for
example, using TRAIL.18,20–22,34–38
Cell Death and Disease
Increased activity of TRAIL against cell cycle-arrested tumor cells
H Ehrhardt et al
8
ALL-54
100
100
80
80
events (%)
events (%)
mock
sicyclinE
mock
sicyclinB
60
40
60
40
20
20
0
0
0
50
100
150
200
propidium iodide
0
250 k
50
100
150
200
propidium iodide
250 k
ALL-54
*
*
specific apoptosis (%)
50
25
0
TRAIL
–
+
mock
–
+
sicyclinB
–
+
sicyclinE
Figure 6 Molecular cell cycle arrest in G1 or G2 promotes apoptosis induction by TRAIL in xenografted ALL cells. (a, b) ALL-54 cells were transiently transfected with
siRNA against cyclinB, cyclinE or a mock sequence using single nucleofection, as described in Materials and Methods. Cells were investigated for cell cycle distribution 24 h
after transfection (a) or were stimulated with TRAIL (10 ng/ml) for another 24 h (b). Determination of cell cycle distribution and apoptosis induction, presentation and analysis of
the data, and statistical analysis were performed as in Figures 1 and 4. *Po0.05
Increased activity of TRAIL against resting tumor cells
might explain why TRAIL is especially effective in combination
with cytostatic drugs, which induce cell cycle arrest. Our data
highlight TRAIL as a promising candidate in contrast to others
for the combination with cytostatic drugs.18,20,37,39 Future
polychemotherapy protocols might position TRAIL in close
relation to cell cycle in order to gain highest antitumor
efficiency by TRAIL, based on the molecular understanding
of drug–drug interactions.
Materials and Methods
Materials. TRAIL was obtained from Pepro Tech (Hamburg, Germany).
Alternatively, trimerized TRAIL was produced as described recently, rendering
identical results.39 Caffeine and L-mimosine were obtained from Calbiochem
(Darmstadt, Germany); all further reagents were obtained from Sigma (St. Louis,
MO, USA).
For flow cytometric analysis, the following antibodies were used: anti-cyclinD1
from BD Biosciences (San Jose, SA, USA) and anti-p-HistoneH3Ser10 from Cell
Signaling Technology (Danvers, MA, USA), and anti-DR4 and anti-DR5 from Alexis
Corp. (Lausen, Switzerland), Alx647-conjugated secondary anti-mouse antibody
was obtained from Invitrogen (Darmstadt, Germany); for western blot: anti-FADD,
Cell Death and Disease
anti-FLIP and anti-XIAP from BD Biosciences, anti-Bcl-xL, anti-Bid, anti-cIAP-1 and
anti-PUMA from Cell Signaling; anti-Bak, anti-Bax, anti-Bcl-2, anti-cIAP-2 and
anti-p53 from Santa Cruz (Santa Cruz, CA, USA); anti-Caspase-10 from
MBL International (Woburn, MA, USA); anti-GAPDH from Thermo Fisher
(Waltham, MA, USA); anti-NOXA from Calbiochem (San Diego, CA, USA), and
anti BIM and anti Caspase-8 from Alexis Corp.
Cell lines, xenograft ALL cells and transfection experiments.
HCT116 p53 þ / þ were obtained from B. Vogelstein (The Johns Hopkins
University School of Medicine, Baltimore, MD, USA). All further cell lines were
obtained from DSMZ (Braunschweig, Germany). For leukemic cell line
experiments, cells were seeded at 0.25 106/ml for stimulations of solid tumor
cells at 0.05 106/ml. Tumor cells were incubated with caffeine and
chemotherapeutic drugs, as indicated in the corresponding figure legends.
Informed consent was obtained from all patients in written form, and studies were
approved by the ethical committee of the medical faculty of the Ludwig Maximilians
University Munich (LMU 068-08) and the Children’s Hospital of the TU Munich
(TU 2115/08). Animal work was approved by the Regierung von Oberbayern
(55.2-1-54-2531-2-07). The xenograft mouse model, patient characteristics and
engraftment, amplification, isolation and standardized procedures of in vitro
stimulation have been described in detail recently.11,25,32
Transfection experiments in HCT116 was performed with lipofectamine 2000
(Life Technologies, Grand Island, NJ, USA) according to the manufacturers’
Increased activity of TRAIL against cell cycle-arrested tumor cells
H Ehrhardt et al
9
instructions. Lentiviral transduction of SHEP cells was described recently.18,24,28
Nucleofection of CEM cells was performed with one million cells per reaction, and of
xenograft ALL cells with five million cells per reaction using Amaxa Nucleofector
technology (Lonza, Cologne, Germany) and program C16.32 Lipofection in HCT116
and nucleofection in CEM cells was performed three times consecutively every 12 h.
Lentiviral transduction was performed annealing the following sense and
corresponding antisense oligonucleotides for the generation of pGreen-Puro
shcyclinB 50 -GTCGGATCCGAAATGTACCCTCCAGAAATTGAATTCGTTTCTGGA
GGGTACATTTCTTTTTAAGCTTAGT-30 and 50 -GATCCAAGTGCTACTGCCGCA
GTATCTTCA AGAGAGATACTGCGGCAGTAGCACTTTTTTTC-30 for the
construct containing shcyclinE.24 DR4 and DR5 cDNAs were obtained from
imaGENES GmbH (Berlin, Germany) and were cloned into pcDNA3.1.39 For the
transient knockdown of cyclinB or cyclinE in HCT116, CEM and xenograft ALL cells,
siRNA against cyclinB (50 -GAAAUGUACCCUCCAGAAAtt-30 , 20 mM) and siRNA
against cyclinE 50 -AAGTGCTACTGCCGCAGTATCtt-30 , 20 mM) were obtained
from MWG Biotech (Ebersberg, Germany). All Star negative control siRNA from
Qiagen (Hilden, Germany) was used for control transfection.
Apoptosis assays, flow cytometric analyses and western blot.
Determination of cell death induction was performed with forward side scatter
analyses for leukemia cells and with Nicoletti staining for solid tumor cells.
Apoptotic cell death was confirmed in selected experiments with Annexin
V–propidium iodide double staining as described.11,18,28 To discriminate between
G2 and M arrest, double staining with p-Histone H3 and propidium iodide was
performed; to separate G0 and G1 phase, double staining with cyclinD1 and
propidium iodide was performed, as described recently.18,25 After cell fixation in
70% ethanol overnight, cells were resuspended in PBS with 0.25% Triton X,
followed by incubation with the specific antibodies overnight in PBS supplemented
with 1% BSA. After three washing steps, cells were incubated with RNAse
A (100 mg/ml) at 37 1C and propidium iodide was added at 10 mg/ml directly before
flow cytometric analysis. For the detection of TRAIL-death receptors, cells were
incubated with the specific primary antibody, followed by incubation with an
anti-mouse IgG1-specific antibody conjugated to Alx647 (Life Technologies).
To exclude dead cells, death receptor staining was followed by Annexin V
(BD Biosciences)/propidium iodide (1 mg/ml) double staining. LSR II
(BD Biosciences) was used for the determination of cell cycle distribution and
TRAIL receptor expression gating on living cells, and data were analyzed using
Flow Jo software version 8.8.6 (Ashland, OR, USA). Western blot analysis was
performed using a lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl,
1 mM Na2 EDTA, 1 mM EGTA, 1% Triton X, 2.5 mM sodium pyrophosphate, 1 mM
beta-glycerophosphate and 1 mM Na3VO4 supplemented with proteinase inhibitor
cocktail set I (Merck, Darmstadt, Germany) according to the manufacturer’s
instructions.
Statistical analysis. Specific apoptosis was calculated as ((apoptosis of
stimulated cells at end minus apoptosis of unstimulated cells at end) divided by
(100 minus apoptosis of unstimulated cells at end) times 100).
All data are presented as the mean values of at least three independent
experiments±S.E.M., unless otherwise stated. Isobolograms were performed with
CompuSyn software version 1.0 (ComboSyn Inc., Paramus, NJ, USA). To test for
significant differences, the paired t-test was applied to compare two groups; for
multivariate analysis, one-way RM ANOVA was used. Statistical significance was
accepted with Po0.05.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements. The skilled technical work of L. Mura is kindly
appreciated. The work was supported by DFG (SFB684, TP22), Deutsche José
Carreras Leukämie Stiftung (R10/26), Bettina Bräu Stiftung and Dr. Helmut
Legerlotz Stiftung (all to I.J.).
1. Li Y, Laterra J. Cancer stem cells: distinct entities or dynamically regulated phenotypes?
Cancer Res 2012; 72: 576–580.
2. Vermeulen L, de Sousa e Melo F, Richel DJ, Medema JP. The developing cancer stem-cell
model: clinical challenges and opportunities. Lancet Oncol 2012; 13: e83–e89.
3. Mordant P, Loriot Y, Lahon B, Castier Y, Lesèche G, Soria JC et al. Minimal
residual disease in solid neoplasia: new frontier or red-herring? Cancer Treat Rev 2012;
38: 101–110.
4. Müller V, Alix-Panabières C, Pantel K. Insights into minimal residual disease in cancer
patients: implications for anti-cancer therapies. Eur J Cancer 2010; 46: 1189–1197.
5. Wiezorek J, Holland P, Graves J. Death receptor agonists as a targeted therapy for cancer.
Clin Cancer Res 2010; 16: 1701–1708.
6. Johnstone RW, Frew AJ, Smyth MJ. The TRAIL apoptotic pathway in cancer onset,
progression and therapy. Nat Rev Cancer 2008; 8: 782–798.
7. Ashkenazi A, Herbst RS. To kill a tumor cell: the potential of proapoptotic receptor agonists.
J Clin Invest 2008; 118: 1979–1990.
8. Amm HM, Oliver PG, Lee CH, Li Y, Buchsbaum DJ. Combined modality therapy with
TRAIL or agonistic death receptor antibodies. Cancer Biol Ther 2011; 11: 431–449.
9. Testa U. TRAIL/TRAIL-R in hematologic malignancies. J Cell Biochem 2010; 110: 21–34.
10. Sayers TJ, Murphy WJ. Combining proteasome inhibition with TNF-related apoptosisinducing ligand (Apo2L/TRAIL) for cancer therapy. Cancer Immunol Immunother 2006; 55:
76–84.
11. Ehrhardt H, Wachter F, Maurer M, Stahnke K, Jeremias I. Important role of Caspase-8 for
chemo-sensitivity of ALL cells. Clin Cancer Res 2011; 17: 7605–7613.
12. Georgakis GV, Li Y, Humphreys R, Andreeff M, O’Brien S, Younes M et al. Activity of
selective fully human agonistic antibodies to the TRAIL death receptors TRAIL-R1 and
TRAIL-R2 in primary and cultured lymphoma cells: induction of apoptosis and
enhancement of doxorubicin-and bortezomib-induced cell death. Br J Haematol 2005;
130: 501–510.
13. Buchsbaum DJ, Zhou T, Grizzle WE, Oliver PG, Hammond CJ, Zhang S et al. Antitumor
efficacy of TRA-8 anti-DR5 monoclonal antibody alone or in combination with
chemotherapy and/or radiation therapy in a human breast cancer model. Clin Cancer
Res 2003; 9: 3731–3741.
14. Wen J, Ramadevi N, Nguyen D, Perkins C, Worthington E, Bhalla K. Antileukemic drugs
increase death receptor 5 levels and enhance Apo-2L-induced apoptosis of human acute
leukemia cells. Blood 2000; 96: 3900–3906.
15. Brooks CL, Gu W. New insights into p53 activation. Cell Res 2010; 20: 614–621.
16. Resnick-Silverman L, Manfredi JJ. Gene-specific mechanisms of p53 transcriptional control
and prospects for cancer therapy. J Cell Biochem 2006; 99: 679–689.
17. Wu GS, Burns TF, McDonald ER 3rd, Jiang W, Meng R, Krantz ID et al. KILLER/DR5
is a DNA damage inducible p53-regulated death receptor gene. Nat Genet 1997; 17:
141–143.
18. Ehrhardt H, Schrembs D, Moritz C, Wachter F, Haldar S, Graubner U et al. Optimized
anti-tumor effects of anthracyclines plus vinca alkaloids using a novel, mechanism-based
application schedule. Blood 2011; 118: 6123–6131.
19. Castro Alves C, Terziyska N, Grunert M, Gündisch S, Graubner U, Quintanilla-Martinez L et al.
Leukemia-initiating cells of patient-derived acute lymphoblastic leukemia xenografts are
sensitive toward TRAIL. Blood 2012; 119: 4224–4227.
20. Jin Z, Dicker DT, El-Deiry WS. Enhanced sensitivity of G1 arrested human cancer cells
suggests a novel therapeutic strategy using a combination of simvastatin and TRAIL. Cell
Cycle 2002; 1: 82–89.
21. Fulda S, Debatin KM. Sensitization for tumor necrosis factor-related apoptosis-inducing
ligand-induced apoptosis by the chemopreventive agent resveratrol. Cancer Res 2004; 64:
337–346.
22. Ray S, Shyam S, Fraizer GC, Almasan A. S-phase checkpoints regulate Apo2 ligand/
TRAIL and CPT-11-induced apoptosis of prostate cancer cells. Mol Cancer Ther 2007; 6:
1368–1378.
23. Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P et al. Impact of
mutant p53 functional properties on TP53 mutation patterns and tumor phenotype:
lessons from recent developments in the IARC TP53 database. Hum Mutat 2007; 28:
622–629.
24. Ehrhardt H, Höfig I, Wachter F, Obexer P, Fulda S, Terziyska N et al. NOXA as critical
mediator for drug combinations in polychemotherapy. Cell Death Dis 2012; 3: e327.
25. Ehrhardt H, Pannert L, Pfeiffer S, Amtmann E, Jeremias I. Enhanced anti-tumor effects of
vinca alkaloids given separately from cytostatic therapies. Br J Pharmacol 2012; 168:
1558–1569; online first.
26. Anastasov N, Klier M, Koch I, Angermeier D, Höfler H, Fend F et al. Efficient shRNA
delivery into B and T lymphoma cells using lentiviral vector-mediated transfer. J Hematop
2009; 2: 9–19.
27. Klier M, Anastasov N, Hermann A, Meindl T, Angermeier D, Raffeld M et al. Specific
lentiviral shRNA-mediated knockdown of cyclin D1 in mantle cell lymphoma has minimal
effects on cell survival and reveals a regulatory circuit with cyclin D2. Leukemia 2008; 22:
2097–2105.
28. Ehrhardt H, Haecker S, Wittmann S, Maurer M, Borkhardt A, Toloczko A et al. Cytotoxic
drug-induced, p53-mediated upregulation of Caspase-8 in tumor cells. Oncogene 2008;
27: 783–793.
29. Greshock J, Nathanson K, Martin AM, Zhang L, Coukos G, Weber BL et al. Cancer cell
lines as genetic models of their parent histology: analyses based on array comparative
genomic hybridization. Cancer Res 2007; 67: 3594–3600.
30. Sandberg R, Ernberg I. Assessment of tumor characteristic gene expression in cell lines
using a tissue similarity index (TSI). Proc Natl Acad Sci USA 2005; 102: 2052–2057.
Cell Death and Disease
Increased activity of TRAIL against cell cycle-arrested tumor cells
H Ehrhardt et al
10
31. Peller S, Rotter V. TP53 in hematologic cancer: low incidence of mutations with significant
clinical relevance. Hum Mutat 2003; 21: 1017–1026.
32. Höfig I, Ehrhardt H, Jeremias I. Efficient RNA interference in patients’ acute
lymphoblastic leukemia cells amplified as xenografts in mice. Cell Commun Signal
2012; 10: 8.
33. Schmitz M, Breithaupt P, Scheidegger N, Cario G, Bonapace L, Meissner B et al.
Xenografts of highly resistant leukemia recapitulate the clonal composition of the
leukemogenic compartment. Blood 2011; 118: 1854–1864.
34. Schlereth K, Charles JP, Bretz AC, Stiewe T. Life or death: p53-induced apoptosis requires
DNA binding cooperativity. Cell Cycle 2010; 9: 4068–4076.
35. Schlereth K, Beinoraviciute-Keller R, Zeitlinger MK, Bretz AC, Sauer M, Charles JP et al.
DNA binding cooperativity of p53 modulates the decision between cell-cycle arrest and
apoptosis. Mol Cell 2010; 38: 356–368.
36. Gonzalves F, Ashkenazi A. New insights into apoptosis signalling by Apo2L/TRAIL.
Oncogene 2010; 29: 4752–4765.
37. Valeriote F, van Putten L. Proliferation-dependent cytotoxicity of anticancer agents: a
review. Cancer Res 1975; 35: 2619–2630.
38. Rixe O, Fojo T. Is cell death a critical end point for anticancer therapies or is cytostasis
sufficient? Clin Cancer Res 2007; 13: 7280–7287.
39. Grunert M, Gottschalk K, Kapahnke J, Gündisch S, Kieser A, Jeremias I. The adaptor
protein FADD and the initiator Caspase-8 mediate activation of NF-kB by TRAIL. Cell
Death Dis 2012; 3: e414.
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